Project Summary/Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the top five deadliest cancers due to the lack of effective treatment options. While PDAC tumor cells are of epithelial identity, mesenchymal cells (mostly fibroblasts) surrounding the tumor also expand during PDAC development and play complex roles in both inhibiting and enabling the tumor. Such paradox is thought to occur at least partly due to the presence of multiple subtypes of cancer associated fibroblasts (CAFs), with some having tumor inhibitory and others having tumor promoting functions. The distinction of those subtypes is to date poorly defined at the molecular and cellular levels, the elucidation of which could lead to development of additional strategies to treat cancer. The long term goal of this project is to determine the cellular origin, molecular identity and functions of CAF subtypes. In preliminary studies, I found that Gata6, a key transcription factor determining mesenchymal cell fate during development, is expressed in a subpopulation of CAFs in both human and mouse PDAC. The central hypothesis of this proposal is that Gata6 positive CAFs, originating from tissue residence fibroblasts, function to restrain PDAC progression. Specific aim 1 will determine the function of Gata6 positive CAFs in PDAC. A genetically engineered mouse model of spontaneous PDAC (Pdx1FlpOki; FSF-KrasG12D/+; p53frt/+ (KPF)) will be interbred with Fsp-cre; Gata6flox/flox mice, which will lead to fibroblast specific deletion of Gata6. Tumor progression and molecular changes in both the tumor and microenvironment will be evaluated. Specific aim 2 will utilize reporter alleles to determine the cellular origin of Gata6 positive CAFs. Three potential cellular sources of fibroblasts will be examined, including tissue resident fibroblasts, bone marrow mesenchymal cells, and pancreatic epithelial cells. My preliminary studies have demonstrated that Isl1creER; Tomatoflox alleles label tissue resident fibroblasts in the normal pancreas. Those alleles will be brought together with the KPF alleles to lineage trace the descendants of tissue resident fibroblasts in PDAC. Secondly, GFP labeled bone marrow will be transplanted into the KPF mice to identify bone marrow derived fibroblasts. Lastly, a GFPfrt reporter allele will be incorporated into the KPF tumor model to label the pancreatic epithelial cells. In these experiments, co-expression of Gata6 and Tomato or GFP reporters will identify the cellular sources of Gata6 expressing CAFs. The sponsor of this grant, Dr. Gustavo Leone, has extensive experience with genetically engineered mouse models of the tumor microenvironment and a track record of training successful postdoctoral fellows. The dynamic and collaborative environment in the Hollings Cancer Center will also ensure the success of the proposed project. In summary, this study will provide novel insights in CAF heterogeneity in PDAC, including new molecular signatures, cellular origins and novel gene functions. Ultimately, such knowledge in the tumor microenvironment may reveal additional targets and therapeutic avenues to inhibit tumor progression and prolong PDAC patient survival.